In Vitro and In Vivo Evaluation of Scaffolds

All the scaffolds we work with are taken through comprehensive evaluation steps to determine their suitability for usage in tissues. This package of scaffold evaluation briefly includes:

Viability:
Cell growth on the scaffolds (different skeletal cells depending on what tissue the scaffold is designed for) over multiple days is assessed by the alamarBlue® metabolic assay.

Morphology:
Fluorescent staining of cells over multiple time points and qualitative assessment of correct morphological appearance. For tendon cells, which appear elongated in situ, we quantify measurements of cell shape (aspect ratio and circularity) (Fig 8)

Gene expression:
In order to determine how the cells are responding to the scaffolds, we carry out gene expression analysis over multiple time points. For bone scaffolds this will be the expression of genes important in the differentiation of bone forming cells (osteoblasts). For tendons it would be genes important for tendon health, as well as genes associated with tendon disease.

Immunogenicity:
To get an indication of the likelihood a scaffold will induce an immune response once implanted, we expose the scaffolds to human pre-macrophage cells and measure their activation by pro-inflammatory cytokine gene expression and secretion.

Bone in vivo model:
To assess the efficacy of a bone scaffold in vivo we create a “critical size defect” (a defect that by itself will not heal) in the skull of a rat, and implant the scaffold into the defect. After 4 and 12 weeks we assess the amount of new bone formed by micro-CT and histological analysis.

Tendon in vivo model:
To assess the efficacy of a tendon scaffold in vivo, we create a tear of the rotator cuff tendon in the shoulder of a rat (one of the most common tendon tears). In this model we remove the tendon from the bone, and reattach it in a manner that mimics the current surgical procedure. In this model we lay the scaffold of interest over the tendon tear and assess healing 6 and 12 weeks later by histological analysis and by testing the biomechanical strength of the repaired shoulder.

See caption.
Figure 8: Human Tendon Cells growth and differentiation on a scaffold. Day 1 demonstrates cell proliferation, Day 3 where the cells are differentiating. Confocal fluorescent micrographs.

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